Fluorescein isothiocyanate covalently to the epsilon amino groups of lysine residues of cytochrome c under basic conditions. Four derivatives have been isolated by ion exchange chromatography, each one having one fluorescein per molecule of cytochrome c. Even though the spectral evidence indicated that the heme environment is unaltered in these derivatives, the kinetic constants for succinate-cytochrome c reductase and cytochrome oxidase activities are different for each derivative. Three derivatives no longer have a high affinity site for cytochrome oxidase. The fourth derivative has a normal Km but lower Vmax with the oxidase, while having an increased Km in the reductase assay. We are currently trying to map the location of the modified lysines for each derivative in order to determine which portions of the cytochrome c molecule are involved in interactions with the oxidase and reductase. After incorporation of these derivatives into submitochondrial particles, the intrinsic fluorescence may be used to monitor internal pH changes. Similarly, the diester of 6-carboxyfluorecein may be used to measure the internal pH in intact mitochondria. The ester derivative diffuses through the mitochondrial membranes, where internal esterases release 6-carboxyfluorescein. Due to its high negative charge (-3), the chromophore is retained within the mitochondria, permitting the internal pH to be monitored. These techniques are being used to evaluate the relationship between transmembrane pH gradients and energy conservation.